N-Ethylcarboxamidoadenosine (12) was synthesised from adenosine (1) and the 6-chloro-2’,3’-O-isopropylidene-AT-ethylcarboxamidoadenosine (25) was synthesised from inosine (19). Employing molecular modelling techniques and the results from previous structure activity relationships it was possible to design and synthesise a N6-substituted N-ethylcarboxamidoadenosines which possessed an oxygen in the N6-substituent either in the form of an epoxide (which was obtained by cpoxidising an alkene with m-CPBA or dimethyldioxirane) or in the form of a cyclic ether as was the case for N6-((tetrahydro-2H--pyran--2-yl)methyl-N-ethylcarboxamidoadenosine (78). These compounds were tested for their biological activity at the A1 adenosine receptor by their ability to inhibit cAMP accumulation in DDT, MF2 cells. The EC50 values obtained indicated that the N6-(norborn-5-en-2-yl)-N-ethylcarboxamidoadenosines were the most potent. Of theseN6-(S-endo-norbrn-5-en-2-yI)-N-ethylcarboxaniidoadenosine (56) was the most potent (0.2 nM). N6-(exo-norborn-5-en-2-yl)-2-iodo-N-ethylcarboxamidoadenosine (79) was synthesised from guanosine (22) and was also evaluated for its potency at the A, receptor (24.8 ± 1.5 nM). At present 79 is being evaluated for its selectivity for the A1 receptor compared to the other three receptor subtypes (A2a, A2b, A3). A series of N6-(benzyl)-N-ethylcarboxamidoadenosines were synthesised with substitutions at the 4-position of the phenyl ring. Another series of compounds were synthesised which replaced the methylene spacer between the N6H and the N6-aromatic or lipophilic substituent The replacement groups -were carbonyl and trans-2- cyclopropyl moieties. The N6-acyl compounds were obtained by reacting 2’,3’-O- di(tert-butyldimethylsilyl)-AT-ethylcarboxamidoadenosinc (59) with the appropriate acid chloride and then deprotecting with lelrabutylammonium fluoride in tetrahydrofuran. The compound N6-(4-(1,2-dihydroxy)ethyl)benzyl-N- ethylcarboxamidoadenosine (125) was synthesised by the reaction of 4-(1,2-0- isopropylidene-ethyl)benzyl aminc (123) with 6-chloro-2,3-0-isopropylidene-N- ethylcarboxamidoadenosine (25). Compound 123 was synthesised from an epoxidation of vinylbenzyl phthalimide (118) followed by an acidic ring opening to yield the diol which was isopropylidenated to yield 4-(l,2-O-isopropylidene- elhyl)benzyl phlhalimide (122), It was hoped that the presence of the diol functionality in 125 would increase water solubility whilst maintaining potency at the A3 receptor.

The serotonin receptor 2A (5-HT₂[A]) is a member of the G-protein coupled receptor family and is of interest due to its role in physiological functions such as smooth muscle contraction, platelet aggregation, thermoregulation, learning and memory. More importantly, 5-HT₂[A] has also been implicated in CNS disorders including schizophrenia, depression and anxiety. A yeast two-hybrid screen had previously been carried out to identify proteins that interacted with 5-HT₂[A] and therefore may modulate intracellular function. The cytoskeletal actin-binding protein filamin-C was identified as a possible 5-HT₂[A] interacting partner. The aim of the research in this thesis was to further investigate the potential interaction between 5-HT₂[A] and filamin-C and to investigate functional roles for the interaction. A fragment of human filamin-C, aa 2162-2725, was shown to interact with the C-terminus of human 5-HT₂[A] using two in vitro techniques, the yeast-two hybrid system and a GST capture assay. The region of filamin-C needed to bind to 5-HT₂[A] was narrowed to the start of repeat 20, aa 2251, through to aa 2424 at the beginning of repeat 22 and comprises 182 residues. The 5-HT₂[A] region needed to bind to filamin-C was ascertained via yeast two-hybrid to be 31 amino acids between 394-423. Work was performed to determine whether FLNC mRNA was expressed in neural and glial cells and whether FLNC and HTR2A mRNA were co-expressed in any cells. FLNC mRNA was identified in seven out of eight neural and glial cell lines and western blot analysis confirmed this finding at the protein level. Two cell lines, U-118MG and A172, were found to contain both HTR2A and FLNC mRNA. Co-immunoprecipitation experiments showed endogenous filamin-C bound to endogenous 5-HT₂[A] and this complex could be precipitated using anti-filamin-C antibody. Additionally, a GST-5-HT₂[A] fusion complex was found to bind to endogenous filamin-C from U-118MG cells. Immunofluorescent labelling of cells was used to study filamin-C and 5-HT₂[A] proteins in vivo. U-118MG cells showed staining for 5-HT₂[A] around the membrane of the cell, as well as in the cytoplasm, whereas filamin-C staining occurred in the cytoplasm. Co-localisation analysis identified some areas of overlap between 5-HT₂[A] and filamin-C in the cytoplasm of U-118MG cells. The functional role for the 5-HT₂[A]/filamin-C colocalisation was investigated. It was postulated that filamin-C may be involved in the internalisation of 5-HT₂[A]. To test this hypothesis, an in vivo model system was used to investigate whether disruption of the filamin-C/5-HT₂[A] interaction affects internalisation of the receptor. The key preliminary findings of this study, which used expression of a competitor peptide, to disrupt and co-interact, suggested that the filamin-C/5-HT₂[A] interaction is not essential for the internalisation of receptors in response to ligand binding. However, this interaction was important for delivery or maintenance of 5-HT₂[A] to the cell membrane, and expression of the competing peptide caused an accumulation of cytoplasmic 5-HT₂[A].

HIV infection and disease is a multistage process that involves a variety of cell types as the virus spreads through the body. Initially, dendritic cells (DCs) present at the mucosal site of infection bind and internalise HIV for degradation and presentation to T cells. As the DCs migrate to lymph nodes and mature, part of the internalised virions remains infective inside endosomal compartments. During formation of the immunological synapse between CD4 T cells and DCs, infective virions from dendritic cells are transferred to CD4 T cells leading to a strong infection of those cells allowing rapid virus dissemination throughout the body and establishment of the typical HIV infection. Various membrane receptors are involved in this process. Initial HIV binding to DCs is mediated by C-type lectin receptors such as the mannose receptor or DC-SIGN (DC specific intracellular adhesion molecule 3 grabbing non integrin) which is followed by virus internalisation and lysis albeit virus induced changes in endocytic routing prevents a proportion from degradation. Productive infection of DCs has also been observed allowing trans infection of CD4 T cells through a different mechanism. HIV infection of CD4 T cells, DCs and other cells is a multistep process initiated by binding of HIV envelope gp120 to the CD4 receptor, a 55 kDa transmembrane glycoprotein. Subsequent conformational changes in gp120 allow binding to a chemokine receptor, either CCR5 or CXCR4, followed by membrane fusion and infection. The aim of this thesis was to investigate protein associations with the HIV receptors DC-SIGN and CD4 in order to elucidate the mechanism of complex formation, virus entry and/or defining target sites for antiretroviral drugs. This thesis used a proteomic approach for studying the receptors with mass spectrometry-based protein identification as its core technology. A range of different approaches were developed and compared for identification of protein interactions and characterisation of the identified protein associations. An affinity purification of the CD4 receptor complex from lymphoid cells was used as the basis for detecting novel CD4-binding proteins. For this approach a strategy based on mass spectrometry identification of CD4 associating proteins using affinity chromatography and affinity-tag mediated purification of tryptic peptides was developed. This method proved successful for the identification of CD4 interacting proteins such as the strongly associated kinase p56lck, however a limited number of non-specifically bound proteins were also identified along the receptor complex. Using one-dimensional SDS-polyacrylamide gel electrophoresis followed by in-gel digests and mass spectrometry analysis, a large number of non-specifically binding proteins were identified along the CD4/lck complex. Evaluation of different lysis buffers in several independent experiments demonstrated that there was a large and inconsistent array of proteins that were obviously non-specifically bound to the receptor. No further specific binding partners were detected. These data suggested that protein interactions of CD4 on this cell type are of weak and/or transient nature. It also demonstrated a need for careful interpretation of proteomic data in the light of the propensity of non-specific binding under these conditions. To overcome dissociation of weak protein interactions, a method was developed using chemical cross-linking to preserve weak protein interactions on lymphoid cells. Affinity purification was used to purify CD4 along with cross-linked associated proteins and mass spectrometry analysis identified an interaction with the transferrin receptor CD71 and the tyrosine phosphatase CD45. The CD45-CD4 interaction is well known. The CD4-CD71 interaction was demonstrated to be a result from colocalization of the two molecules during formation of endocytic vesicles. Flow cytometry-based fluorescence resonance energy transfer (FRET) measurements were applied to confirm colocalization. A similar interaction was suspected for CD4 and DC-SIGN on the plasma membrane of DCs as cis infection of DCs has been demonstrated i.e. initial binding to DC-SIGN then to CD4/CCR5 on the same cell. Therefore, protein associations of DC-SIGN were investigated using the developed techniques. Using cross-linking, DC-SIGN was shown to assemble in large complexes on the surface of immature monocyte-derived DCs. Mass spectrometry analysis of the purified complexes identified them as homo-oligomers of DC-SIGN. The absence of CD4 suggested that the fraction interacting with CD4 at any one time must be small. The complexes of DC-SIGN were further characterised to be tetramers and successfully co-immunoprecipitated with HIV gp120 and mannan. DC-SIGN monomers were not evident demonstrating that the assembly of DC-SIGN into tetramers is required for high affinity binding of its natural and viral ligands. Thus potential antiviral agents aimed at blocking the early stage of HIV binding to DCs must simulate tetramers in order to neutralise the virus efficiently. Overall the thesis provides new information on protein interactions of CD4 and DC-SIGN, a careful investigation of "proteomics" techniques for identifying the proteins in affinity-purified samples and demonstrates the need for multifaceted analytical approaches to probe complex cellular systems.

"January 2002" / Bibliography: leaves 204-230. / vii, 230, [31] leaves : ill. (chiefly col.), plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002

The ability of a cell to respond to extrinsic stimuli critically depends on its ability to regulate specific intracellular protein - protein interactions in a reversible manner and allow the temporal - spatial characteristics of the signal to be accurately transduced to downstream targets. Growth factor and cytokine receptors provide a link by which extracellular stimuli are propagated within the cell to accomplish specific cellular functions. Ligand - stimulation of these receptors activates cascades of intracellular events that transduce the signals that lead to a variety of cellular responses. The specificity of these signals and the fidelity with which they are communicated within the cell are critical for the fate of an organism as deregulation or misbalance of signalling networks is commonly associated with a wide range of pathologies and diseases. Cytokines are important regulatory proteins that regulate diverse cellular functions through their ability to bind to specific cell surface receptors. Most cytokines are pleiotropic effectors that regulate multiple cellular functions. For example, many cytokines can regulate diverse biological activities such as cell survival, proliferation and differentiation and in many cases these different biological activities can be independently regulated. The regulation of pleiotropic biological responses is mediated through the modulation of multiple intracellular signalling pathways. These pathways often present a high level of redundancy in terms of the biological functions that they control. However, pleiotropic cytokines have the ability to independently activate signalling pathways that lead to the regulation of specific biological functions such as survival, proliferation, differentiation or activation. The molecular mechanisms by which cytokines can regulate pleiotropic biological responses from the activation of a limited number, often redundant, of intracellular signalling pathways have not been fully resolved and remains one of the most important unanswered questions in cell biology. In particular, proteins and molecular mechanisms responsible for specifying different biological responses remain largely unidentified. In many cases, activation of multiple signalling pathways and integration of the signals they transduce is needed in order to modulate a biological function. One important mechanism by which signalling pathways are assembled within the cell is through the action of protein scaffolds that contain phosphotyrosine ( e.g. SH2, PTB ) or phosphoserine / threonine ( e.g. 14 - 3 - 3, WW, FHA, PBD, BCRT ) binding modules. Interestingly, although phosphotyrosine and phosphoserine / threonine - dependent signalling pathways are highly integrated within the cell, scaffold proteins containing both phosphotyrosine and phosphoserine or phosphothreonine - binding domains ( i.e. SH2 / PTB and WW / FHA / PBD / BCRT ) have not been identified. The broad aim of this thesis is to study the fundamental molecular mechanisms by which cytokines, through the binding of cell surface receptors, are able to activate and integrate signalling pathways that regulate and specify cellular responses. In particular, these studies examine the role of the 14 - 3 - 3 family of adaptor proteins in the assembly of signalling networks that couple the activated receptors of the haematopoietic cytokines IL - 3, IL - 5, and GM - CSF to downstream signalling targets and specific cellular functions such as survival and proliferation. The specific aims of this thesis are to examine the composition, molecular mechanisms of assembly and functional roles of signalling complexes that use the adaptor or scaffold protein 14 - 3 - 3 and are important for signal transduction in response to GM - CSF. This work shows that the phosphoserine / threonine - binding scaffold protein 14 - 3 - 3 ζ, previously reported to bind to Ser585 of the GM - CSF receptor, undergoes tyrosine phosphorylation. Using a panel of 14 - 3 - 3 ζ mutants a particular tyrosine residue, Tyr179, was found to be critical for the binding of the SH2 domain of Shc, the assembly of a PI3K signalling complex, the activation of the Akt / PKB signalling pathway and the control of cell survival in response to GM - CSF stimulation. Tyr179 of 14 - 3 - 3 ζ was also found to be important for specifying GM - CSF - mediated biological responses as it was found to play an important role in the control of cell survival versus cell proliferation. Furthermore, it was found that 14 - 3 - 3 ζ is able to simultaneously bind to Ser585 of the GM - CSF receptor and recruit Shc and PI3K through Tyr179, thus integrating phosphoserine / threonine and phosphotyrosine / dependent signalling pathways. The findings described in this thesis helped to identify a novel mechanism by which cytokine receptors achieve both integration in signalling and specificity in biological outcomes. The discovery that phosphoserine / threonine - binding proteins ( i.e. 14 - 3 - 3 ) are themselves tyrosine phosphorylated and able to recruit phosphotyrosine - binding molecules provides a new insight into how intracellular signal integration is achieved. Understanding how signal transduction is carried out within the cell is paramount to successful drug development in many therapeutic areas. The new insights in GM - CSF signalling provided by this work may help to successfully develop treatments to target diseases such as asthma, rheumatoid arthritis and leukaemia, where GM - CSF appears to play a pathogenic role. / Thesis (Ph.D.)--School of Medicine, 2006.

Graduation date: 1997

Graduation date: 1995

The erythroblastoma (ErbB) receptor family consists of four members that are implicated in many human cancers. To gain insight into their biological function, we investigated their interacting partners to derive a comprehensive protein interaction network. Using the membrane yeast two-hybrid (MYTH) system we probed the ErbB2, ErbB3, and ErbB4 interaction space and validated a subset of interacting partners using the luminescence-based mammalian interactome mapping (LUMIER) system. The integrated use of these two complementary protein interaction technologies generated high confidence data and identified many novel ErbB binding partners, a subset of which was supported by co-immunoprecipitation in the breast adenocarcinoma cell line Sk-Br-3 including the GPCR GPRC5B, the cysteine protease CAPN1, and WIF1, a secreted protein containing 5 EGF domains that may represent a novel ErbB ligand. Our systematic approach offers an unbiased systems level view that may identify novel drug targets and contribute to therapeutic research.

The erythroblastoma (ErbB) receptor family consists of four members that are implicated in many human cancers. To gain insight into their biological function, we investigated their interacting partners to derive a comprehensive protein interaction network. Using the membrane yeast two-hybrid (MYTH) system we probed the ErbB2, ErbB3, and ErbB4 interaction space and validated a subset of interacting partners using the luminescence-based mammalian interactome mapping (LUMIER) system. The integrated use of these two complementary protein interaction technologies generated high confidence data and identified many novel ErbB binding partners, a subset of which was supported by co-immunoprecipitation in the breast adenocarcinoma cell line Sk-Br-3 including the GPCR GPRC5B, the cysteine protease CAPN1, and WIF1, a secreted protein containing 5 EGF domains that may represent a novel ErbB ligand. Our systematic approach offers an unbiased systems level view that may identify novel drug targets and contribute to therapeutic research.

The normal function of the thyroid hormone (TH) system is essential for growth, development and metabolism in humans as well as in other species. The action of TH is dependent on its binding to thyroid hormone receptors (THR) found in the cell nucleus. In some situations, chemicals with structural similarities to TH can bind to these receptors and disrupt their normal function. It has been previously demonstrated that environmental contaminants including, carbamazapine, nonlyphenol (NP), bisphenol A (BPA), and several others are able to bind to the THR as either agonists or antagonists and modulate downstream biochemical responses. Municipal wastewater effluent (MWWE) is a major source of these contaminants entering aquatic environments. Recently extracts of MWWE have been shown to contain chemicals that are capable of binding to THRs. However, MWWE is a complex mixture of chemicals and the specific chemicals have not been identified. In this thesis, a proof of concept was developed for using an Effects Directed Assessment (EDA) approach to isolate thyroid receptor active compounds in MWWE. An EDA is a technique created to extract and identify chemicals from complex mixtures, using various fractionation methods. Once these chemicals have been identified, they are further reviewed for biological relevance. A competitive binding assay for THR was developed and applied to determine the relative binding affinity of known environmental contaminants to THR. Nuclear thyroid hormone receptors were isolated from rainbow trout liver by differential centrifugation. This method involved liver tissue homogenization and subsequent centrifugations to separate the nuclear fraction containing the receptors. The binding characteristics of the isolated THR were evaluated using the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in a competitive binding assay. Minimal binding affinity was present in this assay and future studies should validate the assay further and assure that it is comparable to literature values. Environmental contaminants, including BPA, NP were also tested to determine their relative binding affinity to the THRs compared to the endogenous hormones. High concentrations of both BPA and NP bound to the thyroid hormone receptor, displacing radiolabeled T3 from its binding site. The rainbow trout competitive binding assay was also used to test the binding affinities of extracts from two municipal wastewater effluents collected in the Grand River watershed in southern Ontario. Effluents were extracted using a solid phase adsorbent (HLB Oasis cartridge), eluted with methanol, taken to dryness then reconstituted in ethanol for use in the assay. Both effluent extracts displaced the binding of radiolabeled T3 to the thyroid receptors. The studies demonstrate that a competitive THR assay can be used to detect chemicals in complex mixtures with the potential to interact with THRs. The next step should be to apply the assay using an EDA approach to isolate and identify specific chemicals in effluents that are not yet known to bind to the THR. Interference with the normal function of the TH system has the potential to disrupt normal growth, development and metabolism in aquatic organisms in the receiving environments.