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
| Identifer | oai:union.ndltd.org:BSU/oai:cardinalscholar.bsu.edu:handle/188469 |
| Date | January 2008 |
| Creators | Shrestha, Mahesh K. |
| Contributors | Vann, Carolyn N. |
| Source Sets | Ball State University |
| Detected Language | English |
| Format | 82 leaves : ill. (some col.) ; 28 cm. |
| Source | Virtual Press |
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