title: Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy.
abstract: Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic β-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in β-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 β-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gβ and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gβ and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gβγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gβγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gβγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in β-cells occurs at the level of G-protein release.
authors: Biomedical Engineering, Vanderbilt University, Nashville, Tennessee.
authors: Brittany
authors: B
authors: Caldwell
authors: Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri.
authors: Alessandro
authors: A
authors: Ustione
authors: Biomedical Engineering, Vanderbilt University, Nashville, Tennessee; Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri. Electronic address: piston@wustl.edu.
authors: David W
authors: DW
authors: Piston
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000378:metabolism*; D050637:Receptors, Dopamine D3 / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
chemical_list: C000600618:DRD2 protein, mouse; C493594:Drd3 protein, mouse; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3