One of the oldest, extant, lineages of vertebrates, the sea lamprey, was used to clarify the evolutionary origin and divergence of the growth hormone receptor (GHR) family. A single, full-length, cDNA, and a second, partial, cDNA were identified and shown to encode proteins that share amino acid identity with GHRs and prolactin receptors (PRLR s) previously identified. The complexity of the dynamic signaling system, with special emphasis on this system in fish and in the context of the evolution of this system, is discussed in the first chapter. The second chapter integrates the new insights gained by these studies. Included is a newly proposed phylogenetic analysis and revised nomenclature-system for vertebrate GHRs that better represents the evolutionary history of the receptor family. The molecular evolution of the receptors is, furthermore, highlighted as the backdrop for the continued discussion regarding how the GH-family of hormones exhibit such coordinated and pleiotropic actions.

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) and is a core component of the neuromuscular junction, facilitating fast synaptic transmission leading to muscle contraction. Mutations to the human adult muscle nAChR lead to various forms of congenital myasthenic syndrome (CMS), a disease characterized by progressive fatigable muscle weakness. A central channel pore constriction formed by a ring of five leucine residues (L9’) forms part of the nAChR channel gate. CMS-causing mutations in the L9’ residues lead to a form of CMS that results in longer channel opening times and a delayed signal decay. To understand better how L9’ mutations in the human adult muscle nAChR influence channel function, I used two-electrode voltage clamp electrophysiology to perform a comprehensive mutant screen of all L9’ residues in each subunit of the human adult muscle nAChR. This resulted in a total of 76 unique mutations: 19 L9’ mutations consisting of every possible natural amino acid substitution in each subunit (α, β, ε, δ). The results of this screen show that while the polarity and size of a substituted residue contribute to its effect on channel function, increasing the polarity of the side chain typically has a more potentiating effect on channel function than does a change in size. The subunit in which the mutation is expressed also tailors the effect of a given mutation on channel function, with several δL9’ mutations producing qualitatively different effects than equivalent mutations in other subunits. Because the majority of L9’ mutations resulted in a gain-of-function, I originally postulated that interactions between L9’ and surrounding residues stabilize the resting state with the elimination of such interaction through mutations destabilizing the resting state to promote channel gating. Using a double mutant cycle, I explored interactions between the L9’ and adjacent non-L9’ residues but found that there are only weak or no interactions that contribute to channel function. Instead, my data support the hypothesis that the nAChR operates via a hydrophobic gating mechanism, and that adjacent L9’ residues are driven together by the hydrophobic effect to form a closed pore. L9’ mutations that either increase the polarity or decrease residue size likely reduce the hydrophobic driving forces that stabilizes the resting state, thus leading to an enhancement in channel function.

No description available.

No description available.

Co-translational targeting of secretory and integral membrane proteins to the endoplasmic reticulum (ER) requires two key mediators, the signal recognition particle (SRP) and its receptor. The SRP receptor is composed of two tightly associated subunits termed SRα and SRβ. Very little is known about the mechanism of membrane assembly of these two subunits of the SRP receptor. Therefore, it is the aim of this thesis to study the interactions between SRα and SRβ on the ER membrane as well as the role of SRα and SRβ in membrane assembly of functional SRP receptor. Unlike typical endoplasmic reticulum (ER) integral membrane proteins, both subunits of the SRP receptor were extracted from the ER membrane with 0.08% deoxycholate; 0.2M Tris pH 9.0. Nevertheless, SRβ could be targeted to the ER only when the SRP dependent pathway of translocation was functional, similar to other integral membrane proteins of the ER. Urea resistant anchoring of SRa on the ER membrane was sensitive to limited digestion of the membranes with trypsin (Andrews eta/., 1989). However, anchoring of SRα was restored by incorporating exogenous SRβ into trypsin treated membranes, confirming that one function of SRβ is anchoring of SRα. Consistent with this is the observation that, SRβ could be immunoprecipitated in a complex with SRα but not with SRα mutants containing deletions in the anchoring domain. Finally, an antiserum to the GTP binding domain of SRβ inhibited translocation of the secretory protein preprolactin suggesting that SRβ also has a direct role in translocation. / Thesis / Master of Science (MSc)

Substantia nigra neurons produce dopamine in response to cholinergic stimuli that may involve receptor operated Ca²⁺ -entry that has been associated with the transient receptor potential (Trp) proteins. There were 6 Trp isoforms reported when I started this work. I set out to determine which isoforms of Trp mRNA were expressed in the substantia nigra using the whole brain for comparison. I initially used RT-PCR to determine the Trp mRNA expression. Subsequently, I used competitive RT-PCR for quantifying the major isoforms. Finally, I confirmed my results by Co-RT-PCR of the major isoforms. Trp3 and Trp6 were found to be the predominant forms expressed in the substantia nigra and whole brain, while the levels of Trps 1, 2, 4 and 5 were very low in both. Estimation of mRNA levels using competitive RT-PCR showed that the Trp6 mRNA levels in substantia nigra and the whole brain were similar while those for Trp3 were significantly lower in the substantia nigra than in the while brain. Thus substantia nigra differs from the whole brain in its Trp expression. Properties of Trps 3 and 6 are not fully known. Trp3 is regulated by IP₃-receptor activation but both Trp 3 and 6 can be activated by diacylglycerol. How this relates to the signal transduction events in substantia nigra remains to be determined. / Thesis / Master of Science (MS)

The regulation of gene expression at the level of transcription is an important mechanism for maintaining homeostasis. The peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear hormone receptor superfamily that is involved in transcriptionally modulating such pathways as lipid and fatty acid metabolism. This receptor binds to enhancer elements, peroxisome proliferator-responsive elements (PPREs), upstream of a variety of target genes including those involved in β-oxidation of fatty acids, in the peroxisome and mitochondria, and ketogenesis. One such element has been identified upstream of the rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase (mtHMG-CoAS) gene. This enzyme has been shown to be one of the key regulatory points of ketogenesis. To learn how PPARα mediated transcriptional regulation occurs, this receptor was used as bait in a yeast dihybrid screen and was found to interact with human mtHMG-CoAS. Reproduction of this interaction 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 was performed by solid phase capture assays, using GST fusion proteins, and by co-immunoprecipitations. It was also ascertained that the synthase enzyme interacts with the retinoid X receptor α (RXRα). The hamster cytoplasmic form of the enzyme was chosen as a control and showed no binding capabilities to either nuclear hormone receptor. Interestingly, the mitochondrial enzyme contains a motif, LXXLL that has previously been shown to be important for binding between a transcriptional coactivator and a receptor. A site-directed mutant of the mitochondrial synthase sequence from LASLL to LASVL was made. The mutant showed a reduced ability to interact with both nuclear receptors. Consequently, the LXXLL motif is responsible, at least in part, for the interaction between mtHMG-CoAS and both PPARα and RXRα. The cytoplasmic synthase does not contain the motif; its corresponding sequence is LASVL. The effect ofthe mitochondrial synthase within a cell was then determined by transient transfection assays. It was discovered that on the HMG PPRE the mitochondrial synthase potentiated PPARα mediated transactivation while on the AOx PPRE the enzyme inhibited it. Thus, mtHMG-CoAS modulates PPARα activity in a PPRE dependent manner. The LXXVL mitochondrial mutant inhibited PPARα transactivation on both the HMG and AOx PPRE. Therefore, the mutant acts as a dominant-negative inhibitor of α mediated activity. The cytoplasmic control enzyme had no effect on either PPRE. To determine if the mitochondrial enzyme could be detected within the nucleus where it appeared to be modulating PPARα mediated transcription, localization studies were performed with the use of immunofluorescence. Immunofluorescence was done by utilizing hemagglutinin (HA) epitope tagged fusions of the mitochondrial, mutant and cytoplasmic enzymes. When the HA tag was placed at the carboxyl terminus of mtHMG-CoAS, the enzyme was localized to the mitochondria with no apparent nuclear staining. This is consistent with previous localization studies done with the rat mtHMG-CoAS. Also, the mutant enzyme, with the HA tag at the carboxyl terminus, was only detected in the mitochondria. However, under certain conditions, when PPARα and the mitochondrial synthase were co-transfected, the mitochondrial enzyme was detected within both the mitochondria and the nucleus. The mutant, on the other hand, when co-transfected with PPARα was found to remain non-nuclear. The HA tagged cytoplasmic control enzyme was also non-nuclear. Therefore, mtHMG-CoAS can be detected within the nucleus, it binds, due to at least in part a LXXLL motif, to nuclear receptors, and it is capable of modulating transcription in a PPRE dependent manner. Ketogenesis becomes an important mechanism for fuel production during starvation, prolonged exercise and diabetes. Perhaps, during extreme circumstances such as starvation, mtHMG-CoAS is involved in autoregulation which allows for an amplification of the transcription of its own gene. Also, mtHMG-CoAS appears to inhibit the transcription of the AOx enzyme which is involved in peroxisomal fatty acid β-oxidation. Again, perhaps under extreme conditions, the β-oxidation of fatty acids is concentrated within the mitochondria which allows for the production of acetyl-CoA that can subsequently be converted to ketone bodies that can then be utilized for fuel. / Thesis / Master of Science (MS)

The multiple physiological effects of the indoleamine hormone melatonin, are mediated primarily by its two G protein-coupled MT1 and MT2 receptors. Our group has shown an upregulation of melatonin receptors following treatment with histone deacetylase (HDAC) inhibitors, including valproic acid (VPA) and Trichostatin A, in cultured cells and/or in the rat brain. VPA increases histone H3 acetylation at the MT1 gene promoter region in rat C6 glioma cells, indicating that this epigenetic mechanism underlies its upregulation of MT1 expression. Since HDAC inhibitors can also alter DNA methylation, the possible involvement of this second major epigenetic mechanism in the regulation of MT1 expression, was examined. C6 cells were treated with the DNA demethylating agent, azacytidine (AZA, 1 - 25 µM), for 24 or 48 hours. Treatment of C6 cells with AZA caused a significant upregulation of MT1 mRNA expression, as compared with controls (DMSO 0.05%). Moreover, treatment with AZA (10 or 20 µM) for 24 or 48 hours, suppressed or abolished DNMT1 protein expression, and inhibited DNMT1 mRNA expression, which indicates inhibition of the DNMT1 enzyme activity. A combination of VPA and AZA caused a trend toward additive upregulation of the MT1 receptor. These results show that DNA demethylation plays a role in the regulation of the MT1 receptor, consistent with the well-known effects of this epigenetic mechanism on gene transcription. Epigenetic regulation of melatonin receptor expression could provide a novel strategy for modulating the therapeutic effects of this hormone and its clinically relevant agonists, such as agomelatine, and could also provide avenues for enhancing the antioxidant, neuroprotective, oncostatic and other benefits of this hormone and its agonists. / Thesis / Master of Science (MSc) / The hormone, melatonin, is involved in maintenance of the sleep cycle, and has many neuroprotective effects, initiated by its binding to specific proteins called receptors. Epigenetic or reversible chemical modifications which alter DNA, without changing its sequence, can alter the levels of these receptors. This process can be modulated by drugs, which can increase levels of the melatonin receptor. In this study, the drug 5-Azacytidine (AZA) was used to cause specific chemical changes to DNA, termed demethylation. This thesis shows for the first time, that AZA causes an increase in melatonin receptors. AZA’s ability to cause demethylation was confirmed by observing decreased levels of the protein responsible for DNA methylation, DNA methyltransferase. Melatonin receptors in the brain exhibit changes in disorders such as Alzheimer’s and Parkinson’s disease. Understanding the mechanisms underlying the regulation of these receptors could provide avenues for enhancing the neuroprotective benefits of melatonin and related drugs.

No description available.

The glycine receptor (GlyR) is a ligand-gated ion channel member of the Cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Zinc is a potent allosteric modulator of GlyR function, enhancing GlyR activity at low nM to 10[mu]M concentrations while inhibiting GlyR activity at higher concentrations. We investigated sources of contaminating zinc, identifying low nM levels of zinc in ultrapure H₂O, powdered reagents used in the preparation of common electrophysiological buffers, and in polystyrene pipets. These low levels of zinc were capable of enhancing GlyR function. These findings suggest that without checking for this effect using a zinc-chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of zinc with drugs of abuse on taurine-activated GlyR to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by drugs of abuse, indicating no synergistic effects on efficacy when receptors are saturated with taurine as may be seen during synaptic events in vivo. Finally, we utilized phage display to identify novel peptide modulators of the GlyR. We tested 26 peptides against [alpha1beta] GlyRs, identifying peptides with various levels of activity on GlyR function. We demonstrated that these modulators were zinc-dependent, as their effects on GlyR activity were abolished in the presence of the zinc-chelating agent tricine. Together, these data indicate the importance of accounting for the effects of zinc when studying the function of the GlyR, as even low levels of zinc that can be found as contaminants in labware and buffers can affect GlyR function and responses to various allosteric modulators, including drugs of abuse. / text