In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs

Sci Rep. 2015 Jun 23;5:11133. doi: 10.1038/srep11133.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques
  • Cell Line, Tumor
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Embryo, Nonmammalian / metabolism
  • Genes, Reporter
  • HEK293 Cells
  • Humans
  • Mice
  • Osteosarcoma / metabolism
  • Protein Binding
  • Protein Interaction Mapping*
  • Receptors, G-Protein-Coupled / metabolism*
  • Saccharomyces cerevisiae / metabolism
  • Xenograft Model Antitumor Assays
  • Zebrafish / embryology
  • Zebrafish / metabolism

Substances

  • Receptors, G-Protein-Coupled
  • Cyclic AMP-Dependent Protein Kinases