Rapid Cell Signaling
G-protein coupled, seven-transmembrane (7-TM) receptors (GPCRs) represent large, diverse gene families found in fungi, plants and animals. The GPCR family of receptors is involved primarily with internalizing extracellular environmental signals via interaction with intracellular G-proteins. GPCR ligands and modulators range from peptide and non-peptide molecules to chemokines, nucleosides, growth factors and light. The involvement of GPCRs in many critical cellular signaling responses has resulted in considerable interest as a pharmacologically important protein family. Furthermore, the ligand binding sites of this 7-TM family are expressed outside of the cell membrane allowing relatively easy access to small molecule drugs making the gene family highly druggable. Binding of ligands or agonists to a GPCR activates the associated trimeric GTP-binding protein (G protein). G proteins are comprised of an α-subunit, which binds guanine nucleotide and hydrolyzes GTP to GDP when activated, and a β- and γ-subunit complex (Figure 1).
Figure 1. Schematic diagram of G-Protein Coupled Receptor (GPCR) Signaling Pathways.
Different subtypes of each G protein subunit can be brought together to achieve diverse functional outputs by activating cell signaling pathways. For example, the activation of the Gas-coupled receptors activates the effector adenylate cyclase (AC) to convert cytosolic ATP to cyclic AMP (cAMP). Conversely, activation of Gai-coupled receptors inactivates AC. Activation of the Gaq sub-family triggers the effector phospholipase C (PLC) to cleave membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts on IP3 receptors found in the membrane of the endoplasmic reticulum to elicit Ca2+ release. Each of these small molecules involved in GPCR activation, cAMP, PIP2, IP3, DAG and Ca2+ are termed second messengers and can be used as a target by researchers to study GPCR function and to provide assays for the discovery of small molecules that can modulate GPCR activity.
A common reagent for quantifying Ca2+ release is the plasma membrane permeable dye Fluo-4 AM. After incubation with live cells, endogenous cellular esterases cleave the acetoxymethyl ester functional groups producing the dye Fluo-4, which becomes highly fluorescent in the presence of Ca2+. Biosensors for second messengers are also commercially available which can be transiently transfected into cells using the viral transfection reagent BacMam 2.0. Of consequence, however with all these assays is the rapid onset and short lifetime of second messengers requiring fast kinetic assays.
To accurately quantify second messengers, assay readout systems must utilize on-board injectors that provide rapid addition of agonists to cells and immediate kinetic detection with a temporal resolution of about one (1) second. While PMT-based microplate readers that measure cell population-based results can be used for these assays, imaging-based readouts allow for cell sub-population analysis where only positively responding cells are counted. This tends to provide significant increases in assay window, in the order of 3-4 fold.
BioTek’s Lionheart FX Automated Microscope has offset injector tips to enable rapid inject/image operations for fast kinetic reactions.
- Detection of Human IgG Using a Luminescence-based Proximity Assay
- Optimization of a Multi-Mode Detection Model for Measuring Real-Time Cellular Respiration and Mitochondrial Function using Fluorescent Biosensors
- Monitoring Protein Dehydrogenase Activity with Filter-based Absorbance Spectroscopy
- Characterizing Gs- and Gi-dependent Regulation of cAMP Levels in Real Time with cADDis Biosensor and Automated Live Cell Imaging
- Automated Imaging Assay for Characterizing Ca2+ Flux with R-GECO Biosensor
- Live Cell Imaging of GPCR Dependent Second-Messenger Systems
- Characterizing Calcium Mobilization using Kinetic Live Cell Imaging
BioTek’s Visual Abstracts are brief animated presentations describing the workflow of a specific application.
- Lionheart FX Automated Microscope
- Cytation 7 Cell Imaging Multi-Mode Reader
- Cytation 5 Cell Imaging Multi-Mode Reader
- Cytation 1 Cell Imaging Multi-Mode Reader
Learn more about Fast Kinetics applications - search our Application Notes and technical documentation in Resources.