Abstract
Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.
Highlights
Most signal transduction pathways transmit receptor-mediated input signals through a relay of intracellular signaling events
Encoded Renilla Luciferase (Rluc) Protein-fragment Complementation Assay (PCA) are applied to quantification and characterization of dynamic protein-protein interactions (PPIs) in real time and in vivo
We tested the impact of distinct perturbations of proteins and/or receptor pathways (GPCRs) on defined molecular interactions using an extended PPI reporter platform
Summary
Most signal transduction pathways transmit receptor-mediated input signals through a relay of intracellular signaling events. Signals are sensed and converted by defined cell surface receptors and are transmitted through cytoplasmic and nuclear effector molecules This leads to signal amplification and uncoupling as well as feed-forward and feed-back regulation, which are based amongst others on oscillations of second messenger levels and on modulations of dynamic protein-protein interactions (PPIs). PKA acts as a compartmentalized signaling hub of multiple signaling cascades and is a central regulator and effector of homeostatic and metabolic control8 It is the classic downstream effector of G protein-coupled receptors (GPCRs) which are among the most common targets of therapeutic drug development. To detect and systematically map changes of PPIs involved in aberrant signal transmission, adaptable assays are needed Such PPI reporters would be valuable for genetic or pharmacological studies in different cell- or organism-based model systems. We illustrate that an advanced PPI reporter platform based on mutually exclusive binary interactions of PKA can be applied to analyze PPI dynamics following activation of endogenous GPCR pathways in different model systems
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