The trace amine–associated receptor 1 (TAAR1) is an intracellular G–protein coupled receptor whose activation by trace amines, catecholamines and amphetamines leads to elevation of cyclic–AMP and activation of protein kinase A (PKA). Recently, the Amara lab discovered that TAAR1 also mediates the activation of the small GTPase, RhoA. TAAR1 is expressed in midbrain dopamine (DA) neurons, including those in the substantia nigra and ventral tegmental area, and thus is positioned to modulate both motor activity and addiction–related plasticity. Due to antibody limitations, however, neither the intracellular membrane localization of TAAR1 nor the site of signaling by this receptor has been clearly demonstrated in neurons.
Dopaminergic neurotransmission is a coordinated process which requires synthesis, packaging, exocytosis, and reuptake of DA. Amphetamine (AMPH) can stimulate TAAR1, which has been shown to downregulate the surface expression of the dopamine transporter, thus decreasing DA reuptake and increasing extracellular DA concentrations. In addition, AMPH and elevation of cAMP decreases the activity of the vesicular monoamine transporter, VMAT2 in neurosecretory pheochromocytoma (PC12) cells, although the mechanism of this regulation remains undefined. The co–expression of TAAR1 and VMAT2 in the DA neuron and PC12 cells suggests that TAAR1 activation may mediate the effects of AMPH/cAMP on VMAT2. Towards understanding the role of TAAR1 in transporter trafficking and function in the DA neuron, this thesis seeks to define the mechanism of AMPH action on TAAR1 signaling and examine the intracellular membrane localization and pathways downstream of TAAR1 activation.
In Chapter I, we used compartment–specific FRET–based sensors to determine the functional subcellular localization of TAAR1. Novel endomembrane targeting constructs were designed and targeting, and functionality was confirmed using standard biochemical techniques and confocal microscopy. Targeted FRET–based sensors for PKA and RhoA activation enabled us to assess TAAR1–mediated responses to AMPH treatment in discrete subcellular compartments. AMPH increased PKA activation in the synaptic vesicle compartment. However, TAAR1–mediated effects of AMPH on RhoA signaling was differentially localized to the Golgi and ER membrane compartments.
In Chapter II, it was hypothesized that PKA activation of TAAR1 may negatively regulate VMAT2. We used midbrain DA neuron cultures and SK–N–SH neuroblastoma cells that express TAAR1 and VMAT2 and release catecholamines as model systems. With CRISPR–Cas9 technology the Amara lab generated TAAR1 knockout SK–N–SH cells that were used to examine the role of TAAR1 in VMAT2 regulation. VMAT2–mediated uptake of radiolabeled DA and serotonin was measured in the presence or absence of drugs that modulate VMAT2 activity. Inhibition of the Gα stimulatory (GαS) G–proteins upstream of PKA activation increased VMAT2 uptake; conversely, stimulation of cAMP decreased VMAT2 activity. Compared to wildtype cells, we found no difference in VMAT2 uptake in TAAR1 knockout cells treated with PKA agonists like dibutryl cAMP and forskolin. These data suggest that VMAT2 uptake is modulated by GαS signaling, cAMP and PKA activation, but does not require TAAR1. Taken together our results show that the cAMP–dependent inhibition of VMAT2 uptake by PKA is not mediated by the TAAR1 receptor.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/31290 |
| Date | 24 July 2018 |
| Creators | Scott, Shane Shakar |
| Contributors | Trinkaus–Randall, Vickery, Amara, Susan G. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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