Insulin resistance together with insufficient insulin secretion leads to the development of type II diabetes mellitus. Glucose-stimulation of insulin secretion has been extensively studied, but other pathways that regulate insulin secretion are not as well understood. I investigated the signaling mechanisms involved in the inhibition of insulin secretion by dopamine, which is synthesized by pancreatic ?-cells and co-secreted with insulin. Previous research has shown that dopamine-inhibition of insulin secretion is mediated primarily by the D3 dopamine receptor (DRD3) even though the DRD2 receptor has been reported to be expressed in ?-cells. To further understand this dichotomy, I investigated the dynamic protein-protein interactions between the dopamine receptor subtypes and their heterotrimeric G-proteins using two-color fluorescence fluctuation spectroscopy (FFS). I characterized the use of two fluorescent proteins, mApple and EGFP, to measure dynamic heteromerization changes with FFS. Furthermore, I showed that to detect proper GPCR signaling, both the G? and G? subunits of the G?? complex must be overexpressed in the cell. Triple transfections of a dopamine receptor and G? and G? subunits each labeled with a different fluorescent protein resulted in plasma membrane localization of all three fluorescent proteins and permitted FFS evaluation of interactions between the dopamine receptor and G?? complex. Upon dopamine stimulation, I measured a decrease in protein-protein interactions between the D3 receptor and G?? complex, indicating activation of the D3 receptor. In contrast, no significant changes in protein interactions were measured between the D2 receptor and G?? complex after dopamine treatment. These results demonstrate that two-color FFS is a powerful tool to measure dynamic protein interactions in living cells, and show that preferential DRD3 signaling in ?-cells occurs at the level of G-protein release.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03212016-145439 |
| Date | 04 April 2016 |
| Creators | Caldwell, Brittany Catherine |
| Contributors | Hassane Mchaourab, David Piston, John Gore, Melissa Skala, Anne Kenworthy |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03212016-145439/ |
| Rights | restrictsix, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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