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In diabetes the body lacks the mechanism for producing insulin. This disease is one of the most prevalent in the world, causing a tremendous loss of health, life and economy. Thus, there is a need for developing novel therapies effective in control of diabetes. In an effort to develop such a therapy we have targeted G-protein coupled receptors (GPCRs) to stimulate 13-cells for insulin secretion. GPCRs are membrane bound receptors which respond to a variety of external signals and mediate intracellular signal Stransduction. GPCRs, therefore, are the targets of many current therapeutic drugs. The objective of this study was to generate chimeric receptors containing portions of two closely related GPCRs to identify domains important in binding various ligands to stimulate increased secretion of insulin by f3-cells of the pancreas. In this collaborative research with Kelly Wilbur of Eli Lilly, domains of receptors GPR40 and GPR41 were exchanged at different regions to construct two chimeric receptors (GPR40.431_41.459 and GPR40.567 41.547) using two separate cloning steps to insert these fragments sequentially into the cloning vector, pcDNA3.1. Construction of the chimeric receptors was carefully planned to include specific amino acid residues important in ligand binding. Priority was given to locate the joining section of the two receptor portions at the transmembrane region and to maintain full length of the receptor. This was to maintain the integrity of external and internal loops of the receptors important in ligand binding and signal transduction. Following transformation of the chimeras into E. coli to obtain sufficient DNA, construction of the desired chimeric receptors was verified by agarose gel electrophoresis for size and by PCR for the presence of the correct portions of each receptor. The two constructs were sent to Eli Lilly for sequencing. One construct was found to be appropriately constructed (GPR40.431_GPR41.459) but the other one was unstable and had undergone recombination as is often seen in cloned membrane proteins which can be toxic to E. coli. In the future, Human Embryonic Kidney cells will be transfected with the chimeric receptor and a FLIPR analysis will be performed to assess the activity of the receptor when stimulated by ligands of interest to Eli Lilly. Construction of additional chimeras will be needed in the future to fully understand the specific regions responsible for ligand binding and activation of GPR40 to aid in the design of drugs to stimulate insulin secretion by 03-cells. / Department of Biology

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Communication is a ubiquitous component of life. While complexity and sophistication vary, both unicellular and multicellular organisms constantly interact with their environment. Unicellular organisms, once thought to be asocial, have since been demonstrated to display a multitude of social interactions and hierarchies. For example, quorum sensing enables a bacterial population to modulate gene expression in response to cell-population density, initiating social behavior and the exchange of resources. In eukaryotes, unicellular ascomycete fungi use mating GPCRs to detect secreted peptide pheromones, initiating changes in gene expression required for mating. An overview of communication in unicellular organisms is presented in Chapter 1. In general, these communication systems are characterized by a high degree of fidelity, and as such have been harvested by synthetic biologists to organize communication in synthetic systems. Quorum sensing modules have been employed for pattern formation and to coordinate biosynthesis processes across a community. However, fungal mating remains underutilized as a source of synthetic biology tools. In this dissertation, we leverage fungal mating G-protein-coupled receptors (GPCRs) and their peptide ligands to build responsive yeast systems. We use genome-mining to identify additional fungal peptide-GPCR pairs, which are then characterized in the yeast Saccharomyces cerevisiae. In Chapter 2, we exploit the high specificity and sensitivity of fungal mating GPCRs to design a yeast whole-cell biosensor that produces a visible output in response to detection of peptide biomarkers. In Chapter 3, we genome-mine additional peptide-GPCR pairs and use them as orthogonal signaling channels to build synthetic yeast communities. Finally, in Chapter 4, we use these synthetic yeast communities to provide sense-and-respond capabilities to an Engineered Living Material (ELM).

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∼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.

published_or_final_version / Surgery / Master / Master of Philosophy