Introduction and Objectives: The PDGFβ receptor and its ligand, PDGF-BB, are expressed throughout the central nervous system (CNS), including the hippocampas. Several reports confirm that PDGFβ receptors are neuroprotective against N-methyl-D-asparate (NMDA)-induced cell death in hippocampal neurons. NMDA receptor dysfunction is important for the expression of many symptoms of mental health disorders such as schizophrenia. The serotonin (5-HT) type 7 receptor was the most recent of the 5-HT receptor family to be identified and cloned. 5-HT receptors interact with several signaling systems in the CNS including receptors activated by the excitatory neurotransmitter glutamate such as the NMDA receptor. Although there is extensive interest in targeting the 5-HT7 receptor with novel therapeutic compounds, the function and signaling properties of 5-HT7 receptors in neurons remains poorly characterized. Methods: The SH-SY5Y neuroblastoma cell line, primary hippocampal cultures, and hippocampal slices were treated with 5-HT7 receptor agonists and antagonists. Western blotting was used to measure PDGFß receptor expression and phosphorylation as well as NMDA receptor subunit expression and phosphorylation levels. Real-time RT-PCR was used to measure mRNA level of PDGFß receptor in neuronal cultures. Cell death assays (MAP2, MTT) were used to measure the neuroprotective effects of 5-HT7 and PDGFß receptor activation. Results: My research involved elucidating the molecular mechanisms of neuroprotection after 5-HT7-induced PDGFß receptor upregulation. I demonstrated that 24 h treatment with the selective 5-HT7 receptor agonist, LP 12, increased not only the expression but also the activation of PDGFß receptors as measured by the phosphorylation of tyrosine 1021, the phospholipase Cγ binding site. Activation of the 5-HT7 receptor also selectively changed the expression and phosphorylation state of the NR2B subunit of the NMDA receptor. Activation of 5-HT7 receptors was neuroprotective against NMDA-induced toxicity in primary hippocampal neurons and this effect required PDGFß receptor kinase activity. Thus, long-term (24 h) activation of 5-HT7 receptors was neuroprotective via increasing the expression of a negative regulator of NMDA activity, the PDGFß receptor. In contrast, acute activation (5-30 min) of 5-HT7 receptor increased NMDA evoked current and altered NMDA receptor subunit phosphorylation in hippocampal neurons in a manner that was different from what we observed in our 24 h experiments. Conclusions: I identified two 5-HT7 receptor to NMDA receptor pathways: acute activation of the receptor increased NMDA-evoked currents whereas long-term 5-HT7 agonist treatment prevented NMDA-induced excitotoxicity in a PDGFß receptor-dependent manner. This research is significant in the ongoing advances for the treatment of mental heath disorders, such as schizophrenia and depression, that involve the 5-HT, glutamate, and neuronal growth factor systems.

Thesis (Master, Biology) -- Queen's University, 2009-09-24 14:35:26.369 / A major challenge in neurobiology is to understand the control of behaviour at the molecular level. Neuropeptides and their receptors offer promising candidates for the regulation of various behaviours and changes in physiology. Neuropeptides act as important signaling molecules in the central and peripheral nervous system; they are involved in development, reproduction and metabolism. Most of the neuropeptides and hormone protein receptors belong to the large super family of G-protein coupled receptors. NPR-14 is one of the GPCRs in C. elegans, similar in sequence to the mammalian orexin and cholecystokinin receptors which have a primary involvement in food stimulation, locomotion and local search behaviour on food, egg laying and stimulation of wakefulness and energy expenditure. In this study we determined the expression pattern of npr-14 and characterized the behavioural phenotypes associated with NPR-14 receptor in C. elegans included. We showed that the NPR-14 receptor exhibits the regulation of roaming behaviour and fat in the presence of food in a manner resembling that of a similar receptor NPR-9. Additionally, the NPR-14 receptor is involved in the control of some other behaviours such as: egg-laying, crawling and thrashing but not in the regulation of mechanosensation responses and defecation. In our neuronal expression studies the npr-14 promoter fused to reporter mCherry was found to be expressed in ASH or ASI sensory neurons and DD and VD motor neurons and also VC motor neurons that extend to the vulva region to modulate reproduction and egg-laying. Moreover, based on our studies of NLP-5 and NLP-6 neuropeptides we speculated that these neuropeptides and especially NLP-6 could be the putative ligand for NPR-14 receptor. We also compared the phenotypes associated with the receptor NPR-15. NPR-15 is more similar to the Drosophila tachykinin receptor which has no known function in Drosophila. NPR-15 is unrelated in sequence to NPR-9 and NPR-14 and was thus used as a control. Indeed, we were able to show that NPR-15 appears to regulate responses to posterior mechanical stimulation and rhythmic defecation behaviour in C. elegans. These are phenotypes not associated with NPR-9 or NPR-14. / Master

Title page, summary and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / The insulin and insulin-like growth factor receptor (IR and IGF-lR respectively) networks are ancient and fundamental systems that control growth and metabolism in multicellular organisms. This thesis has examined several aspects of this field focusing on mammalian receptor biology and a comparison of the similarities and differences between the insulin and IGF receptor signalling systems. The insulin receptor family of proteins consist of eleven structural domains, of which the extracellular domains contain all the ligand binding and specificity determinants. The insert domain, within the extracellular region is the least understood of all the domains, and it has no similarity to any other protein sequence. It does however contain the cleavage site which separates the receptor into two subunits and also a small stretch of residues shown to directly contact bound ligand and which is absolutely required for ligand binding in short recombinant forms of the receptor. In addition, the human insulin receptor, expressed as one of two isoforms, A and B, results in the exclusion or inclusion of 12 amino acids directly adjacent to the ligand contacting amino acids in the insert domain. The A isoform lacking exon11 is expressed ubiquitously and the B isoform containing exon11 is co-expressed mainly in the traditional insulin responsive tissues of liver, muscle, adipocytes and kidney, where it is the dominant isoform. In this thesis recombinant insert domain was expressed in a bacterial system in an attempt to purify folded protein suitable for NMR structural analysis. The results of the expression studies indicated that the insert domain was unstructured in isolation and was unable to be adequately refolded by all conditions tried, although hydrophobic conditions appeared to partially stabilize the structure. The overall conclusions of this project were that the Insert domain is likely to have limited structure, and probably buried within the receptor, and therefore requires the presence of the rest of the extracellular domains to adopt its correct structure. A comparison of the ligand binding and phsophorylation potential between the two human isoforms of the insulin receptor was made. A competition binding assay using europium labelled insulin was developed, that found that both IGF-l and IGF-2 had an increased affinity for the hIR-A, but insulin had a slightly reduced affinity. These results differ from the established literature in the raw values, however the relative ratios of binding strength are consistent. The most likely reason for this is that the europium labelled insulin has a different mode of binding the receptors due to the location of the europium chelate. Interestingly, using europium labelled IGF-l produced results nearly identical to those of conventional competition assays. Phosphorylation assays indicated that the hIR-B isoform was more responsive than hIR-A. Even though IGF-2 and IGF-l had improved affinity for hIR-A, the level of phosphorylation was not as high. The ability of each growth factor to promote cellular proliferation correlated well with the relative strength of binding and activation of the receptor. The regions of the IR and IGF-1R involved in binding substrates and regulators are predominantly found in the juxtamembrane domain and the C-terminal domain, which contain several potential tyrosine and serine phosphorylation target sequences. In this study the effect of mutations in unique tyrosine residues and other residues in the C-terminal domain of the hIGF-lR was investigated. Results of time-course phosphorylation assays showed that mutation of Tyrosine¹²⁵¹ to phenylalanine caused hyperphosphorylation of the receptor and increased proliferation, which was caused by deregulation of a tyrosine phosphatase. A Tyrosine¹²⁵⁰ to phenylalanine mutation had altered kinetics of phosphorylation, displaying an unchanging rate of phosphorylation over time after ligand stimulation. However, proliferation was unaltered, indicating that even under extended exposure to ligand, the initial strength of receptor activation is more critical to affecting the biological response. The Caenorhabditis elegans insulin-like peptide family is a very large family consisting of possibly 38 peptides likely to be both agonists and antagonists of Daf-2 Receptor (IR homologue) signalling. Comparative modelling of all 38 peptides was performed based on the known structures of mammalian peptides. The overall results indicated that good quality models of ins peptides could be made despite the low sequence similarity with the templates. This suggested that it is the conformational shape of the molecule allowable by the individual residues that is most important when modelling and not having a perfect sequence match. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1200443 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

Title page, summary and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / The insulin and insulin-like growth factor receptor (IR and IGF-lR respectively) networks are ancient and fundamental systems that control growth and metabolism in multicellular organisms. This thesis has examined several aspects of this field focusing on mammalian receptor biology and a comparison of the similarities and differences between the insulin and IGF receptor signalling systems. The insulin receptor family of proteins consist of eleven structural domains, of which the extracellular domains contain all the ligand binding and specificity determinants. The insert domain, within the extracellular region is the least understood of all the domains, and it has no similarity to any other protein sequence. It does however contain the cleavage site which separates the receptor into two subunits and also a small stretch of residues shown to directly contact bound ligand and which is absolutely required for ligand binding in short recombinant forms of the receptor. In addition, the human insulin receptor, expressed as one of two isoforms, A and B, results in the exclusion or inclusion of 12 amino acids directly adjacent to the ligand contacting amino acids in the insert domain. The A isoform lacking exon11 is expressed ubiquitously and the B isoform containing exon11 is co-expressed mainly in the traditional insulin responsive tissues of liver, muscle, adipocytes and kidney, where it is the dominant isoform. In this thesis recombinant insert domain was expressed in a bacterial system in an attempt to purify folded protein suitable for NMR structural analysis. The results of the expression studies indicated that the insert domain was unstructured in isolation and was unable to be adequately refolded by all conditions tried, although hydrophobic conditions appeared to partially stabilize the structure. The overall conclusions of this project were that the Insert domain is likely to have limited structure, and probably buried within the receptor, and therefore requires the presence of the rest of the extracellular domains to adopt its correct structure. A comparison of the ligand binding and phsophorylation potential between the two human isoforms of the insulin receptor was made. A competition binding assay using europium labelled insulin was developed, that found that both IGF-l and IGF-2 had an increased affinity for the hIR-A, but insulin had a slightly reduced affinity. These results differ from the established literature in the raw values, however the relative ratios of binding strength are consistent. The most likely reason for this is that the europium labelled insulin has a different mode of binding the receptors due to the location of the europium chelate. Interestingly, using europium labelled IGF-l produced results nearly identical to those of conventional competition assays. Phosphorylation assays indicated that the hIR-B isoform was more responsive than hIR-A. Even though IGF-2 and IGF-l had improved affinity for hIR-A, the level of phosphorylation was not as high. The ability of each growth factor to promote cellular proliferation correlated well with the relative strength of binding and activation of the receptor. The regions of the IR and IGF-1R involved in binding substrates and regulators are predominantly found in the juxtamembrane domain and the C-terminal domain, which contain several potential tyrosine and serine phosphorylation target sequences. In this study the effect of mutations in unique tyrosine residues and other residues in the C-terminal domain of the hIGF-lR was investigated. Results of time-course phosphorylation assays showed that mutation of Tyrosine¹²⁵¹ to phenylalanine caused hyperphosphorylation of the receptor and increased proliferation, which was caused by deregulation of a tyrosine phosphatase. A Tyrosine¹²⁵⁰ to phenylalanine mutation had altered kinetics of phosphorylation, displaying an unchanging rate of phosphorylation over time after ligand stimulation. However, proliferation was unaltered, indicating that even under extended exposure to ligand, the initial strength of receptor activation is more critical to affecting the biological response. The Caenorhabditis elegans insulin-like peptide family is a very large family consisting of possibly 38 peptides likely to be both agonists and antagonists of Daf-2 Receptor (IR homologue) signalling. Comparative modelling of all 38 peptides was performed based on the known structures of mammalian peptides. The overall results indicated that good quality models of ins peptides could be made despite the low sequence similarity with the templates. This suggested that it is the conformational shape of the molecule allowable by the individual residues that is most important when modelling and not having a perfect sequence match. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1200443 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

No description available.

Estrogen and glucocorticoid receptors (ER and GR) are both members of the same subfamily of steroid nuclear receptors, and can both signal classically as ligand-activated transcription factors. However, many responses to estrogen and glucocorticoid exposure occur through the non-classical pathways, which include rapid activation of kinase cascades, activation of membrane-associated receptors, gene regulation through transcription by non-classical transcription factors, and protein regulation by translation and post-translational modification. Male territorial aggression is a hypothalamically-mediated steroid hormone-dependent adaptive behavior in mice. The hypothalamus, which expresses multiple ERs and GRs, is also responsive to estrogen and glucocorticoid treatment at a cellular level. Experiments were conducted to test the effects of estrogen and glucocorticoid interactions on spinogenesis in the ventromedial hypothalamus (VMH) and on male territorial aggression through the resident-intruder paradigm. Studies in male postnatal primary hypothalamic cell cultures demonstrate the expression of classic ERα, the variant ERα-36, and GPR30. PSD-95 protein, a marker for dendritic spines, is increased in response to 12 hours of treatment with the GPR30 agonist G-1 in an ERK/MAPK-dependent manner. Further work in immortalized embryonic hypothalamic cell lines (mHypoE-11 and mHypoE-42) demonstrate non-classical effects of a membrane-limited glucocorticoid on rapid nuclear translocation of the intracellular GR. Additionally, pharmacological inhibition of the ERK/MAPK pathway results in similar GR translocation in the absence of a ligand. Male postnatal primary hypothalamic cell cultures also respond to glucocorticoid exposure with increased 17Î_-E synthesis, suggesting crosstalk between GR signaling and estrogen signaling. Spine density in the gonadally intact adult male VMH decreases following suppression of estrogen synthesis with the aromatase inhibitor letrozole, suggesting estrogen is necessary to maintain spine density. In vivo studies in adult male mice demonstrate that estrogen is necessary to maintain basal peripheral CORT synthesis. Behavior testing using the resident-intruder paradigm showed that dexamethasone-suppression of adrenal CORT synthesis increases the amount of time resident mice spent engaged in aggressive bouts, and CORT treatment 20 minutes prior to aggression testing abolished this effect. The findings presented here provide support for the importance of the interactions between classical and non-classical estrogen and glucocorticoid signaling pathways on hypothalamic spinogenesis and male territorial aggression. / 1 / Jennifer Rainville

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Regulation of the human delta opioid receptor (hDOR) is implicated in the development of tolerance to chronic morphine (Zhu et al., 1999). In addition, DORs are promising analgesic targets for the management of chronic pain states such as inflammatory or neuropathic pain (Cahill et al., 2007). Therefore, in this study, we investigated multiple aspects of hDOR regulation, including receptor phosphorylation, beta-arrestin binding, receptor internalization, down-regulation and desensitization, using recombinant Chinese hamster ovary (CHO) cells expressing the wild-type or various mutant hDOR constructs. We found that structurally diverse delta opioid agonists regulate the hDOR by different mechanisms. We demonstrate that morphine is able to activate the initial step of the regulatory events, phosphorylation of S363, but due to requirements for simultaneous activation of multiple sites, morphine fails to promote beta-arrestin binding, receptor internalization and down-regulation. We also report that peptide delta opioid receptor agonists and a non-peptide agonist SNC80 differ in their ability to down-regulate the hDOR. Further differences in receptor phosphorylation, desensitization and beta-arrestin translocation between these two classes of full DOR agonists are reveled by truncation of the receptor's C-terminus or by mutation of the primary phosphorylation site, S363. Studies using the mutant receptors identify the C-terminus as the important domain for hDOR phosphorylation, beta-arrestin binding and down-regulation by both peptide and non-peptide agonists. S363 within the C-terminus is critically involved in receptor phosphorylation, desensitization and down-regulation, but not in beta-arrestin binding and receptor internalization. In contrast to peptide agonists, SNC80 is able to phosphorylate and activate secondary intracellular domain(s), in addition to the C-terminus, which participate in beta-arrestin recruitment and receptor desensitization and down-regulation. Therefore, agonist-specific differences were detected for multiple regulatory events between morphine, peptide agonists and SNC80. Differential agonist-mediated regulation of the human delta opioid receptor may be used to design pain therapy drugs with improved analgesic properties and minimal side effects.

Thesis (Ph.D.). / Written for the Dept. of Pharmacology and Therapeutics. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 106-117