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Title page for ETD etd-03212016-145439


Type of Document Dissertation
Author Caldwell, Brittany Catherine
URN etd-03212016-145439
Title Dopamine Regulation of Insulin Secretion Investigated by Fluorescence Fluctuation Spectroscopy
Degree PhD
Department Biomedical Engineering
Advisory Committee
Advisor Name Title
David Piston Committee Chair
Anne Kenworthy Committee Member
Hassane Mchaourab Committee Member
John Gore Committee Member
Melissa Skala Committee Member
Keywords
  • Single Molecule Imaging
  • Insulin Secretion
  • Fluorescence Fluctuation Spectroscopy
  • Fluorescence
  • Pancreatic Beta Cells
Date of Defense 2016-01-27
Availability unrestricted
Abstract
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 beta-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 beta-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-protein beta and G-protein gamma subunits of the G-protein beta gamma complex must be overexpressed in the cell. Triple transfections of a dopamine receptor and G-protein beta and G-protein gamma 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 protein beta gamma complex. Upon dopamine stimulation, I measured a decrease in protein-protein interactions between the D3 receptor and G-protein beta gamma complex, indicating activation of the D3 receptor. In contrast, no significant changes in protein interactions were measured between the D2 receptor and G-protein beta gamma 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 beta-cells occurs at the level of G-protein release.
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