Abstract
The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.
Highlights
The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain
In order to engineer a light-activated GPCR for the visualization of receptor trafficking and intracellular Ca2+ signals we introduced the red fluorescent protein mCherry into the III intracellular loop of the light-activated GPCR melanopsin from mouse
Expression of CaMello in Human embryonic kidney (HEK) tsA201 cells and illumination of the cell with red light reveals the localization of the GPCR at the plasma membrane by the intracellular mCh tag
Summary
The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Changes in the intracellular Ca2+ concentration in neurons regulate various cellular processes including synaptic transmitter release, gene transcription, and various forms of synaptic plasticity[1]. These Ca2+ signals are spatio-temporally controlled in their amplitude and can occur as fast Ca2+ spikes or Ca2+ oscillations[2]. Many Ca2+ signaling molecules are assembled into macromolecular complexes in specific subcellular microdomains, which function autonomously within highly specialized environments[1] An example for such a functional subcellular microdomain is the assembly of voltage gated Ca2+ channels with the transmitter release machinery at the presynaptic terminal[3]. Activation of mGluR1 in neurons produces a single Ca2+ transient, whereas mGluR5 generates an oscillatory Ca2+ wave[1,6]
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