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.

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

In this study, a novel method based on biotinylated antibodies and streptavidin coated magnetic beads was used to separate the thrombocyte subpopulations from zebrafish whole blood. DiI-C18, a lipophilic dye, labels only young thrombocytes when used at low concentrations. Commercially available biotinylated anti-Cy3 antibody was used to label the chromophore of DiI-C18 on the young thrombocytes and streptavidin coated magnetic beads were added subsequently, to separate young thrombocytes. The remaining blood cells were probed with custom-made biotinylated anti-GPIIb antibody and streptavidin magnetic beads to separate them from other cells. Further, thrombocytes are equivalents of mammalian platelets. Platelets play a crucial role in thrombus formation. The G-protein coupled receptors (GPCRs) present on the platelet surface are involved during platelet activation and aggregation processes. So, thrombocytes were studied for the presence of GPCRs. The GPCR mRNA transcripts expressed in the young and mature thrombocytes were subjected to densitometry analysis and pixel intensities of the bands were compared using one way ANOVA. This analysis did not show significant differences between the young and mature GPCR mRNA transcripts but identified a novel GPCR, GPR18 that was not reported in platelets earlier. To study the function of this GPCR, it was knocked down using GPR18 specific antisense morpholino and vivo morpholino. The immunofluorescence experiment indicated the presence of GPR18 on thrombocytes. The results of the assays, such as, time to occlusion (TTO) and time to aggregation (TTA) in response to N-arachidonyl glycine (NAG) as an agonist, showed prolongation of time in GPR18 larval and adult morphants respectively, suggesting that GPR18 plays a role in thrombus formation in zebrafish. In conclusion, our results indicate that GPR18 may be present in zebrafish thrombocytes, it may be involved in thrombus formation and that NAG may be an agonist at GPR18 on thrombocytes.

No description available.

The prostaglandins comprise a group of bioactive lipids generated from arachidonic acid by cyclooxygenases and cell type-specific prostaglandin and thromboxane synthases. Prostaglandins mediate local cell signaling interactions by activation of G-protein coupled prostanoid receptors. Because the prostaglandins and their receptors are active in all tissues, they have an extraordinarily broad spectrum of physiological and pathophysiological functions that have hampered the development of safe prostanoid-based medications. This situation has emphasized the importance of understanding the functional properties of the prostanoid receptors and developing selective ligands capable of being used in patient care.The aims of this project were to identify novel regulatory functions of endogenous EP and FP prostanoid receptors in cultured human cells. Our results show that activation of EP₂ receptors in human microglia and astrocytes led to increased secretion of BDNF, a growth factor that regulates the survival of neurons. In the same cell lines, FP receptors regulate the induction of TNF-&alpha; gene expression through a classic G_q-PKC pathway. In microglia these FP receptors also stimulate a novel signaling crosstalk mechanism involving the up-regulation of TCF transcriptional function by Raf kinases, which culminates in the expression of the angiogenic inducer Cyr61. FP receptors also regulate the induction of angiogenic immediate early genes in cultured ciliary muscle cells, which may constitute the early steps in a mechanism by which commercial FP agonists reduce intraocular pressure in glaucoma therapy.The up-regulation of BDNF through glial EP₂ receptors constitutes a mechanism by which elevated PGE₂ in the inflamed brain might elicit either healing processes in the brain or neuronal apoptosis. On the other hand, induction of TNF-&alpha; and Cyr61 by glial FP receptors may mediate neuroinflammation and may also contribute to glioma tumor growth. Stimulation of FP receptors in the ciliary muscle leads to the induction of immediate early genes capable of coordinating tissue remodeling processes that have been previously documented. The results of these studies suggest novel regulatory functions of the prostanoid receptors in the brain and eye. Furthermore, these findings provide insight on how the selective modulation of the EP₂ and FP receptors might be therapeutically advantageous.

Lau Pui Ngan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 171-181). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Abbreviations --- p.viii / Publications Based on work in this thesis --- p.xii / Chapter Chapter 1 --- Introduction and project overview --- p.1 / Chapter 1.1 --- Ghrelin structure and its synthesis --- p.3 / Chapter 1.2 --- Types of growth hormone secretagogues (GHSs) --- p.6 / Chapter 1.3 --- Characterization of GHS-R --- p.7 / Chapter 1.3.1 --- Cloning of GHS-Rla and GHS-Rlb --- p.7 / Chapter 1.3.1.1 --- GHS-R subtypes --- p.7 / Chapter 1.3.1.2 --- Properties of GHS-R subtypes --- p.7 / Chapter 1.3.1.3 --- Evidence of non-GHS-Rla stimulated by ghrelin and GHSs --- p.9 / Chapter 1.3.1.4 --- Distribution of GHS-R --- p.10 / Chapter 1.3.2 --- Signal transduction pathways of GHS-R --- p.11 / Chapter 1.3.3 --- Comparison between human and seabream GHS-R --- p.12 / Chapter 1.4 --- Is adenosine a partial agonist at GHS-Rla? --- p.15 / Chapter 1.5 --- Physiological effects of ghrelin --- p.17 / Chapter 1.6 --- Apoptosis --- p.19 / Chapter 1.6.1 --- Introduction --- p.19 / Chapter 1.6.2 --- Apoptosis versus necrosis --- p.19 / Chapter 1.6.3 --- Mechanisms of apoptosis --- p.20 / Chapter 1.6.4 --- Methods to study apoptosis --- p.23 / Chapter 1.6.5 --- Different types of apoptotic inducers --- p.24 / Chapter 1.7 --- Apoptotic and anti-apoptotic pathways regulated by GPCRs --- p.27 / Chapter 1.7.1 --- Bcl-2 family pathway --- p.27 / Chapter 1.7.2 --- Caspase pathway --- p.27 / Chapter 1.7.3 --- ERK pathway --- p.28 / Chapter 1.7.4 --- PI3K/Akt pathway --- p.29 / Chapter Chapter 2 --- Materials and solutions --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.2 --- "Culture medium, buffer and solutions" --- p.37 / Chapter 2.2.1 --- Culture medium --- p.37 / Chapter 2.2.2 --- Buffers --- p.37 / Chapter 2.2.3 --- Solutions --- p.38 / Chapter Chapter 3 --- Methods --- p.41 / Chapter 3.1 --- Maintenance of cell lines --- p.41 / Chapter 3.1.1 --- Human Embryonic kidney (HEK293) cells --- p.41 / Chapter 3.1.2 --- HEK293 cells stably expressing black seabream growth hormone secretagogues receptors (HEK-sbGHS-Rla and HEK-sbGHS-Rlb) --- p.41 / Chapter 3.2 --- Preparation of plasmid DNA --- p.42 / Chapter 3.2.1 --- Preparation of competent E. coli --- p.42 / Chapter 3.2.2 --- Transformation of DNA into competent cells --- p.42 / Chapter 3.2.3 --- Small-scale and large-scale plasmid DNA preparation --- p.43 / Chapter 3.2.4 --- Confirmation of the purity and the identity of the plasmid DNA --- p.43 / Chapter 3.3 --- Transient transfection of mammalian cells --- p.45 / Chapter 3.4 --- Development of stable cell lines --- p.46 / Chapter 3.4.1 --- Determination of the optimum concentration of each antibiotic used in selection of clones --- p.46 / Chapter 3.4.2 --- Development of monoclonal stable cell line --- p.46 / Chapter 3.4.3 --- Confirmation the expression of 2myc-hGHS-Rla and myc-hGHS-Rlb --- p.48 / Chapter 3.5 --- Measurement of phospbolipase C activity --- p.49 / Chapter 3.5.1 --- Introduction --- p.49 / Chapter 3.5.2 --- Preparation of columns --- p.49 / Chapter 3.5.3 --- [3 H]-inositol phosphate assay --- p.49 / Chapter 3.5.4 --- Measurement of [3H]-inositol phosphates production --- p.50 / Chapter 3.5.5 --- Data analysis --- p.50 / Chapter 3.6 --- Determination of transient transfection efficiency --- p.51 / Chapter 3.7 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.52 / Chapter 3.7.1 --- RNA extraction and first strand cDNA production --- p.52 / Chapter 3.7.2 --- PCR and visualization of amplicons --- p.52 / Chapter 3.7.3 --- Real-time PCR --- p.59 / Chapter 3.7.3.1 --- Construction of standard curve --- p.60 / Chapter 3.7.3.2 --- Data analysis --- p.60 / Chapter 3.8 --- Measurement of caspase-3 activity --- p.65 / Chapter 3.8.1 --- Determination of caspase-3 activity using colorimetric assay --- p.65 / Chapter 3.8.1.1 --- Introduction --- p.65 / Chapter 3.8.1.2 --- Induction of apoptosis --- p.65 / Chapter 3.8.1.3 --- Preparation of cell lysates --- p.65 / Chapter 3.8.1.4 --- Quantification of caspase-3 activity by measuring pNA absorbance --- p.66 / Chapter 3.8.1.5 --- Data analysis --- p.67 / Chapter 3.8.2 --- Determination of caspase-3 activity using bioluminescence resonance energy transfer (BRET2) assay --- p.67 / Chapter 3.8.2.1 --- Introduction --- p.67 / Chapter 3.8.2.2 --- Quantification of caspase-3 activity using BRET2 assay --- p.68 / Chapter 3.8.2.3 --- Data analysis --- p.69 / Chapter 3.8.3 --- Determination of caspase-3 activity using fluorescence resonance energy transfer (FERT) assay --- p.70 / Chapter 3.8.3.1 --- Introduction --- p.70 / Chapter 3.8.3.2 --- Quantification of caspase-3 activity using FRET assay --- p.70 / Chapter 3.8.3.3 --- Data analysis --- p.71 / Chapter Chapter 4 --- Results --- p.72 / Chapter 4.1 --- Characterization of GHS-R --- p.72 / Chapter 4.1.1 --- Properties of GHS-Rla --- p.72 / Chapter 4.1.1.1 --- Constitutively active receptor --- p.72 / Chapter 4.1.1.2 --- Characterization of epitope-tagged hGHS-Rla --- p.73 / Chapter 4.1.2 --- Properties of GHS-Rlb --- p.75 / Chapter 4.1.3 --- Conclusions --- p.75 / Chapter 4.2 --- Effect of co-transfection of HEK293 cells --- p.85 / Chapter 4.2.1 --- Effect of balancing DNA concentrations transfected into HEK293 cells --- p.85 / Chapter 4.2.2 --- Effect of balancing DNA concentration using another Gq-coupled receptor --- p.87 / Chapter 4.2.3 --- Effect of Gi- and Gs-coupled receptor on GHS-Rla signaling --- p.88 / Chapter 4.2.4 --- Potentiating effect of co-transfection appeared using different transfection reagents --- p.88 / Chapter 4.2.5 --- Co-transfection improves transfection efficiency --- p.89 / Chapter 4.2.6 --- Discussions --- p.91 / Chapter 4.3 --- Development of cell lines stably expressing hGHS-Rla or hGHS-Rlb --- p.102 / Chapter 4.3.1 --- Advantages of using a monoclonal cell line --- p.102 / Chapter 4.3.2 --- Sensitivity of HEK293 cells to antibiotics --- p.102 / Chapter 4.3.3 --- Production of polyclonal stable cell line --- p.103 / Chapter 4.3.4 --- Monoclonal stable cell line selection --- p.104 / Chapter 4.3.5 --- Discussions --- p.105 / Chapter 4.4 --- Effect of adenosine on GHS-Rla signaling --- p.111 / Chapter 4.4.1 --- Adenosine acts as partial agonist --- p.111 / Chapter 4.4.2 --- Effect of substance P analog on adenosine-mediated GHS-Rla signaling --- p.112 / Chapter 4.4.3 --- Effect of adenosine deaminase (ADA) on adenosine- and ghrelin-stimulated GHS-Rla signaling --- p.113 / Chapter 4.4.4 --- Specificity of ADA --- p.115 / Chapter 4.4.5 --- Conclusions --- p.116 / Chapter 4.5 --- Role of GHS-R in apoptosis --- p.124 / Chapter 4.5.1 --- Different methods to measure caspase-3 activity --- p.124 / Chapter 4.5.1.1 --- Colorimetric assay --- p.124 / Chapter 4.5.1.1.1 --- Time course for staurosporine and etoposide in HEK293 cells --- p.125 / Chapter 4.5.1.1.2 --- Effect of 2myc-hGHS-Rla on staurosporine- and etoposide-induced caspase-3 activity --- p.127 / Chapter 4.5.1.1.3 --- Time course for staurosporine and etoposide in sbGHS-R monoclonal stable cell line --- p.128 / Chapter 4.5.1.1.4 --- Effect of sbGHS-Rs on staurosporine- and etoposide- induced caspase-3 activityin HEK 293 cells --- p.129 / Chapter 4.5.1.1.5 --- Effect of sbGHS-Rs on staurosporine- induced caspase-3 activity in sbGHS-R monoclonal stable cell line --- p.130 / Chapter 4.5.1.1.6 --- Differences between epitope-tagged and non-tagged sbGHS-Rs in staurosporine- induced caspase-3 activity --- p.131 / Chapter 4.5.1.1.7 --- The role of epitope-tagged sbGHS-Rlbin staurosporine-induced caspase-3 activity --- p.132 / Chapter 4.5.1.1.8 --- Effect of staurosporine and etoposide on GHS-Rla signaling --- p.133 / Chapter 4.5.1.2 --- BRET2 assay --- p.135 / Chapter 4.5.1.3 --- FRET assay --- p.136 / Chapter 4.5.1.4 --- Conclusions --- p.136 / Chapter 4.6 --- Determination of GHS-R amount in terms of mRNA --- p.155 / Chapter 4.6.1 --- Determination of GHS-R amount in stable cell lines --- p.155 / Chapter 4.6.2 --- Transfected DNA amount match with stable cell lines --- p.155 / Chapter Chapter 5 --- "Discussion, Conclusions and Future Plan" --- p.159 / Chapter 5.1 --- General Discussion and Conclusions --- p.159 / Chapter 5.2 --- Future Plan and Experimental Design --- p.168 / References --- p.171

In a phage binding assay, phage clone (3p5A190) expressing a surrogate mas ligand displayed punctate binding and were internalized in cell expressing native mas and GFP-tagged variants. However, the number of bound and internalized phages in cells expressing mas-GFP was substantially less than the cells expressing mas-(Gly10Ser5)GFP and native mas. In parallel, biotinylation experiment quantitatively showed that the extent of mas-(Gly10Ser 5)-GFP translocation was higher than that of mas-GFP. Consistently, cells expressing mas-(Gly10Ser5)-GFP and native mas showed a rapid and sustained increase of intracellular calcium levels upon MBP7 stimulation. By contrast, cells expressing mas-GFP only response to higher concentration of MBP7 challenge and showed a delayed increase of intracellular calcium level. Moreover, cells expressing native mas had a higher proportion (80%) of cells responsive to MBP7 stimulation; in contrast to only 10&sim;20% of cells expressing mas fusion proteins. / MBP7-like motif was identified in human facilitative GLUT1 and GLUT7 indicating that mas might interact with glucose transporter (GLUT) and regulate cellular glucose uptake. GLUT4 was found to be expressed endogenously in the CHO cell by RT-PCR, but expression of insulin receptor was not detectable. Although no statistical difference was detected in basal glucose uptake among control cells Vc0M80 and cells with different levels of mas expression, cells expressing mas-(Gly10Ser5)-GFP showed a high glucose uptake in response to insulin. Furthermore, basal 2-DOG uptake in Mc0M80 cells was not affected by pretreatment with various kinase inhibitors or transient expression of Rho variants. By contrast, MBP7 was found to induce a significant elevation of glucose uptake specifically in Mc0M80 cells transiently transfected with GLUT1. / Orphan G protein-coupled receptor (GPCR) mas was initially isolated from a human epidermal carcinoma. Previous study from our lab identified a surrogate ligand---MBP7 (mas binding peptide 7) for mas, and suggested that GFP tagging might affect the receptor activity of mas. In this project, three stable CHO cell lines expressing native mas, mas-GFP and mas-(Gly10Ser 5)-GFP were used to characterize receptor activity of mas. / To summarize, direct GFP tagging at the C-terminus of mas decreased its interactions with ligand and downstream signaling molecules. Partial recovery of mas receptor activity by adding a peptide linker was confirmed by phage binding, membrane fusion protein translocation and calcium response. In addition, mas was possibily coupled with GLUT1 to affect cellular glucose uptake via signaling pathways yet to be fully characterized. / Sun, Jingxin. / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0104. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 150-170). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Animals rely on the integration of a variety of external cues to understand and respond appropriately to their environment. The relative amounts of food and constitutively secreted pheromone detected by the nematode C. elegans determines how it will develop and grow. Starvation conditions cause the animal to enter a protective stage, termed dauer. Dauer animals are non-eating, long-lived and stress resistant. Yet, when these animals are introduced to food replete conditions they will recover from dauer and proceed into normal development. Furthermore, food restriction has been demonstrated to extend the lifespan of a wide-range of species including C. elegans. However, the exact mechanism by which food signals are detected and transduced by C. elegans to influence development and longevity remains unknown. Here, we identify a G protein-coupled receptor (GPCR) DCAR-1 that acts in two chemosensory neurons to mediate food signaling in an autophagy-related manner. The DCAR-1 ligand Dihydrocaffeic acid (DHCA) competes with dauer-inducing pheromone to promote growth. DHCA is a key intermediate in the shikimate pathway, which is required to synthesize folate and aromatic amino acids. We report that dcar-1 mutations influence dauer formation and extend wildtype lifespan via a mechanism of dietary restriction. Moreover, we show that the lifespan extension of dcar-1 mutants is completely dependent on autophagy gene atg- 18. Furthermore, our data suggests metabolites derived from shikimate are food signals that control aging and dauer development through GPCR signaling in C. elegans. These studies will contribute to the delineation of mechanisms behind the beneficial effects of dietary restriction in eukaryotic organisms. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

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.

Little is known about the outward trafficking of receptor dimers from the endoplasmic reticulum to the plasma membrane, or the role that trafficking plays in assembly, targeting and specificity of receptor signalling. Bimolecular fluorescence complementation was used to follow prescribed receptor homo/heterodimers in Jurkat cells and clarify the trafficking itineraries those receptors follow to reach the plasma membrane. Chemokine receptors CXCR4 and CCR5 were chosen due to their implication in numerous pathologies including, HIV and cancer, and their ability to form homo and hetero-oligomers. This study demonstrates that although the individual receptors composing heterodimeric complexes are the same as in homodimeric complexes, the heterodimer traffics and signals independently of its constituent homodimers. The presence of CD4 affects the trafficking of CCR5 containing dimers but not the CXCR4 homodimer. These observations demonstrate the importance of considering receptor heterodimers as distinct signalling entities that should be more carefully and individually characterized.