Dopamine Regulation of Insulin Secretion Investigated by Fluorescence Fluctuation Spectroscopy
Caldwell, Brittany Catherine
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.