Abstract
Glucose homeostasis is maintained by small clusters of hormone secreting cells in the pancreas: the pancreatic islets. Insulin secreting β-cells make up ∼ 80% of the mouse islet and secrete insulin in a tightly regulated manner. Understanding the mechanisms that regulate insulin secretion is a key factor in developing drugs and therapies for type-2 diabetes and metabolic syndrome.The pancreatic β-cells synthesize dopamine from the circulating precursor L-dopa. We have shown that during glucose stimulated insulin secretion, co-secreted dopamine acts as an autocrine negative regulator of insulin secretion. It does so by activating the dopamine receptor D3 (DRD3) – a member of the G-protein coupled receptor family. DRD3 are present on β-cell plasma membrane, and upon dopamine binding, they attenuate intracellular Ca2+ dynamics. In fact, the frequency of the [Ca2+]i oscillations triggered by 8 mM glucose is diminished by dopamine; these effects are blocked when a selective DRD3 antagonist is added [Ustione and Piston, Mol. Endo. 2012].We investigate the molecular mechanism downstream of DRD3 activation that leads to the changes in intracellular Ca2+ dynamics and insulin secretion. We propose that dopamine activation of DRD3 directly affects β-cell calcium influx via Gβγ complex interaction with the L-type Ca2+ channel (CaV1.2). We test this hypothesis using FRET and two photon excitation FCCS experiments on βTC-3 cells with fluorescent protein labeled versions of the Gβγ complex, and of the CaV1.2 subunit α1C. The goal is to detect the dopamine triggered interaction between the Gβγ complex and the CaV1.2. We can modulate the FRET signal by using the photo-switchable version of the red fluorescent protein rsTagRFP, therefore we can measure FRET from the small percentage of interacting proteins, even in the presence of significant background signal arising from the majority of labeled but noninteracting proteins.
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