No description available.

Assay technologies for GPCRs and their associated G-proteins are in demand for drug screening and other biotechnology applications such as biosensors for diagnostic purposes or odorant/flavour assessment as well as for elucidating the remaining controversial mechanisms in G-protein mediated signalling. This study aims to make progress towards developing a TR-FRET assay for G-protein interactions that could be used as a generic assay platform for GPCR signalling that would be fluorescent, homogeneous and amenable to miniaturization. The first chapter of this study investigates the use of small molecule labels, CS124-DTPA-EMCH:Tb and Alexa546 in a TR-FRET assay. This TR-FRET pair had previously been applied to Gα, Gβγ and RGS4 proteins and during the characterization of this assay, the protein CrV2 was observed to interact with the G-protein. Using TR-FRET, it was demonstrated that a high affinity interaction appears to occur between Gαi1 and CrV2 (Apparent Kd 6.2 nM). CrV2 is encoded by a polydnavirus from endoparasitoid wasps, which is thought to mediate immune suppression, and the interaction with Gα could have important implications in the regulation of the immune system of invertebrates. Improvements to the labelling strategy used in this assay are then attempted through the creation of various G-protein subunit fusions with small, genetically encoded lanthanide binding tags (LBTs) or tetracysteine motifs (TCMs) for site-specific labelling with terbium or FlAsH, respectively. The consequence of the fusions on maintaining G-protein subunit integrity and on the affinity of the tags for their labels is characterized, and then the utility of these constructs as TR-FRET partners is demonstrated. TCM:FlAsH complexes could successfully be used as TR-FRET acceptors for CS124-DTPA-EMCH:Tb labelled binding partners. The interaction between Gβγ2-TCM:FlAsH and Gα:Tb could be measured using TR-FRET and generated an apparent Kd of 3.6 nM. Likewise, LBT:Tb complexes could be used as TR-FRET donors to Alexa546 labelled binding partners which was demonstrated using the chimeric, promiscuous Gα subunit, LBT2:Tb-GαS25 and Gβγ:Alexa. Furthermore, the two site-specific labelling strategies can be used together in TR-FRET studies and an interaction between LBT2:Tb-Gα[subscript]S25 and Gβγ₂-TCM:FlAsH was shown to have an apparent Kd of 2.3 nM. The TRFRET assays were further validated using protease treatments and the addition of unlabelled binding partners reduced the TR-FRET signal. Finally, the feasibility of fusing lanthanide binding tags to GPCRs for alternate assay platforms or other applications was investigated. The β₂- adrenergic and M₂-muscarinic receptors were fused to LBTs and the integrity of the receptors determined using ligand binding and [[superscript]35 S]GTPγS signalling assays. Terbium binding to the LBT was then demonstrated. The utility of these constructs in alternative TR-FRET platforms with Gproteins was then explored. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363937 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

L'objectif principal de ma thèse était de développer de nouvelles technologies et des outils pour l’étude de l’activation des récepteurs couplés aux protéines G (GPCR).À la surface de la cellule se trouve une multitude de récepteurs qui jouent un rôle critique dans la communication cellule-cellule, dont les GPCR, une famille de récepteur utilisant les protéines G intracellulaires pour transmettre leurs signaux. Le ciblage de ces récepteurs à des fins thérapeutique est innovant et très prometteur. Mais à ce jour seuls quelques médicaments ciblant les GPCR ont été mis sur le marché, en partie en raison d'un manque d'outils permettant le suivi de leur action sur les cellules natives.L’objectif de cette thèse est donc de développer des tests simples pour suivre l’activation de n’importe quel GPCR. Pour développer ce type de test, nous avons décidé d'utiliser des fragments d'anticorps appelés nanobodies. Les anticorps sont des protéines du sang produites en réponse à un antigène spécifique qui sont capable de le neutraliser. Les nanobodies correspondent au domaine variable de certains anticorps de camélidés. En raison de leur faible taille (13 kDa) et de leur site de liaison à l'antigène réduit, les nanobodies se lient souvent à des cavités et présentent une grande sensibilité aux changements de conformation de l'antigène. / The main objective of my thesis was to develop technologies and tools to study activation of G protein-coupled receptors (GPCRs).The cell surface is displaying a multitude of receptors, who play critical roles in cell-cell communication. Among them, GPCRs represent a large family relying on the use of intracellular G proteins for their signaling. Targeting these receptors for therapies is very promising and innovative. So far, only few new drugs have been put on the market, partly due to a lack of tools enabling the follow-up of their action on native cells.The aim of this thesis is thus to develop simple assays to study activation of any GPCRs. To develop this kind of test, we used antibody fragments called nanobodies. Antibodies are blood protein produced in response to and counteracting a specific antigen. Nanobodies correspond to antibody fragments derived from the variable domain of a special class of camelid antibodies. Because of their small size (13 kDa) and reduced antigen binding site, nanobodies often bind cavities and show a high sensitivity to antigen conformational changes.

Abstract The genomic organization and mRNA expression of the human GNAL gene on chromosome 18p11.2, a region that has been associated with bipolar disorder and schizophrenia, was determined. The GNAL gene was shown to span over 80 kb and consist of twelve exons, and its structure was very similar to adenyl cyclase stimulating G protein GSα. The start site of transcription was revealed by 5'-RACE. Two polyadenylation signals were found, and 3'-RACE assay was used to verify the functional site. The GNAL gene was expressed as approximately 6 kb transcripts in various regions of the human brain, and no alternative splicing was detected. One informative CA-dinucleotide repeat of 11 alleles and 74% heterozygosity was found in intron 5, and two single nucleotide polymorphisms in introns 3 and 10 were detected by SSCP. During characterization of the GNAL gene, two previously unknown genes were found. A novel intronless gene C18orf2 coding for a functionally unknown protein was localized to intron 5 of the GNAL gene. By semiquatitative RT-PCR, C18orf2 mRNA was found to be moderately expressed in all tissues studied here. Another novel gene, metallophosphoesterase MPPE1, was found to reside adjacent to the 3'-end of the GNAL gene in a tail-to-tail orientation. The deduced amino acid sequence revealed a highly conserved metallophosphoesterase motif gDxH..(16-60)..GDxxdr..(13-34)..GNH[DE], which is typical for various phosphate hydrolyzing enzymes, especially serine/threonine protein phosphatases. The MPPE1 gene contained fourteen exons and spanned about 27 kb. MPPE1 was expressed as a single mRNA of 2.2 kb in various regions of the human brain but not in any other tissues. Four different alternatively spliced forms of MPPE1 were detected by RT-PCR, and each transcript was shown to partially overlap with the 3'-untranslated region of the GNAL gene.

No description available.

The hypothalamic peptide arginine vasopressin (AVP) is an important regulator of adrenocorticotropin (ACTH) release from the anterior pituitary. AVP stimulates ACTH secretion from corticotroph cells by activating the pituitary vasopressin receptor (V1b-R), a member of the G protein-coupled receptor (GPCR) family. In vitro, repeated stimulus of anterior pituitary cells with AVP results in rapid desensitisation. The aim of this research was to develop methods needed to use RNA interference (RNAi) to investigate the role of G protein-coupled receptor kinase 5 (GRK5) in this desensitisation process. This required the development of a model system using human embryonic kidney (HEK) 293 cells transfected with the pituitary vasopressin receptor, V1b-R. AVP binding to the V1bR activates the phosphoinositide signalling pathway, leading to production of inositol phosphates (IPs), which can be measured following radiolabelling of cells with myo-[³H]inositol. Stimulation of V1b-R-transfected cells for 15 min with AVP (100nM) increased IP production to 235.5 ± 23.4 % (n=3, p<0.02) of that seen in un-stimulated control cells. Following a 5 minute pre-treatment with 5nM VP, the IP response to stimulation with 100nM VP for 15 min was reduced to 62.8 ± 9.1 % (n=4, p<0.02) of that seen in control cells that were not pre-treated. These data indicate that AVP-desensitisation can be induced and measured in V1bR-transfected HEK293 cells following a brief pre-treatment with a physiological concentration of AVP. This model system will enable RNAi to be used to investigate the role of GRK5 in AVP-desensitisation. When using RNAi, it is essential to establish that the effects observed are the result of small interfering RNA (siRNA) specific degradation of the target mRNA. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of GRK5 at the mRNA level in HEK293 cells. Human GRK5 mRNA was amplified using qRT-PCR with GRK5 specific primers, providing confirmation that GRK5 is expressed endogenously in HEK293 cells. GRK5 expression studies were carried out to evaluate whether the qRT-PCR methods developed would be suitable to measure knockdown of GRK5 mRNA using RNAi. These experiments were also designed to assess the impact of HEK293 cell culture methods on expression of GRK5. Expression of GRK5 did not vary with passage number (2-26 passages). The GRK5 expression in HEK293 cells that were maintained in culture for 5 days (grown to a confluence of approximately 100%) was 7.4 ± 0.9 fold greater (n=2, p<0.05) than for cells cultured for 3 days (grown to a confluence of approximately 65%). These data indicate that GRK5 expression is affected by HEK293 culture conditions. Furthermore, the results demonstrated that a significant difference in GRK5 expression could be measured in HEK293 cells using qRT-PCR. Therefore the results reported in this thesis provide the basis for future studies utilising RNAi to investigate mechanisms underlying V1b-R desensitisation.

No description available.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.