Rapid Cell Signaling

The 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 nonpeptide 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. This accessability makes 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).

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 G_as-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 G_aq 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. These injectors provide rapid addition of agonists to cells and immediate kinetic detection with a temporal resolution of about one second. While PMT-based microplate readers that measure cell population-based results can be used for these assays, imaging-based readouts allow for cell subpopulation analysis where only positively responding cells are counted. This tends to provide significant increases in assay window, on the order of 3 to 4 fold.

Calcium Kinetics

Kinetic Characterization of Gs- and Gi-Dependent Regulation of cAMP