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Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods
Paula A Dos Santos Claro1, Carolina Inda2, Natalia G Armando3
1Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society, Buenos Aires, Argentina.
Methods in Cell Biology
|January 9, 2019
Summary
Live-cell sensors reveal how Corticotropin-releasing hormone receptor 1 (CRHR1) signaling dynamics, specifically cyclic adenosine monophosphate (cAMP) generation, vary with cell context and receptor location, impacting stress responses.
Area of Science:
- Cellular signaling dynamics
- GPCR signaling pathways
- Stress pathophysiology
Background:
- Classical biochemical methods lack spatial and temporal resolution for studying cellular signaling.
- G protein-coupled receptors (GPCRs) activate downstream signaling, such as cyclic adenosine monophosphate (cAMP) generation.
- Corticotropin-releasing hormone receptor 1 (CRHR1) is implicated in stress and its signaling characteristics are not fully understood.
Purpose of the Study:
- To investigate the real-time, cell context-dependent signaling dynamics of CRHR1.
- To explore the link between CRHR1 activity, cAMP generation, and receptor localization.
- To establish protocols for studying CRHR1 signaling using live-cell biosensors.
Main Methods:
- Utilized Förster resonance energy transfer (FRET)-based biosensors for real-time cAMP and calcium signaling measurements.
- Employed live-cell imaging and analysis techniques.
- Applied fluorescence flow cytometry to quantify cell surface receptor levels.
Main Results:
- CRHR1-elicited cAMP responses exhibit cell context-dependent temporal characteristics.
- A specific link was identified between cAMP generation and CRHR1 signaling originating from internal cellular compartments.
- Protocols for FRET biosensor application in CRHR1 research were detailed.
Conclusions:
- Live-cell biosensor technology provides critical insights into the spatiotemporal dynamics of CRHR1 signaling.
- CRHR1 signaling complexity involves both cell surface and intracellular compartments, influencing cAMP dynamics.
- Understanding these dynamics is crucial for elucidating CRHR1's role in stress pathophysiology.
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