Dopamine Regulation of Insulin Secretion Investigated by Fluorescence Fluctuation Spectroscopy

• Main Author: Caldwell, Brittany Catherine
• Other Authors: Hassane Mchaourab
• Format: Others
• Language: en
• Published: VANDERBILT 2016
• Subjects: Biomedical Engineering
• Online Access: http://etd.library.vanderbilt.edu/available/etd-03212016-145439/

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.